Resonance Raman Characterization of Soluble Guanylate Cyclase Expressed from Baculovirus (original) (raw)
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Interactions of soluble guanylate cyclase with diatomics as probed by resonance Raman spectroscopy
Journal of Inorganic Biochemistry, 2005
Soluble guanylate cyclase (sGC, EC 4.6.1.2) acts as a sensor for nitric oxide (NO), but is also activated by carbon monoxide in the presence of an allosteric modulator. Resonance Raman studies on the structure-function relations of sGC are reviewed with a focus on the CO-adduct in the presence and absence of allosteric modulator, YC-1, and substrate analogues. It is demonstrated that the sGC isolated from bovine lung contains one species with a five-coordinate (5c) ferrous high-spin heme with the Fe-His stretching mode at 204 cm À1 , but its CO adduct yields two species with different conformations about the heme pocket with the Fe-CO stretching (m Fe-CO) mode at 473 and 489 cm À1 , both of which are His-and CO-coordinated 6c ferrous adducts. Addition of YC-1 to it changes their population and further addition of GTP yields one kind of 6c (m Fe-CO = 489 cm À1) in addition to 5c CO-adduct (m Fe-CO = 521 cm À1). Under this condition the enzymatic activity becomes nearly the same level as that of NO adduct. Addition of c-S-GTP yields the same effect as GTP does but cGMP and GDP gives much less effects. Unexpectedly, ATP cancels the effects of GTP. The structural meaning of these spectroscopic observations is discussed in detail.
Biochemistry, 2005
Resonance Raman (RR) spectra of soluble guanylate cyclase (sGC) reported by five independent research groups have been classified as two types: sGC 1 and sGC 2. Here we demonstrate that the RR spectra of sGC isolated from bovine lung contain only sGC 2 while both species are observed in the spectra of the CO-bound form (CO-sGC). The relative populations of the two forms were altered from an initial composition in which the CO-sGC 2 form predominated, with the Fe-CO (ν Fe-CO) and CO stretching modes (ν CO) at 472 and 1985 cm-1 , respectively, to a composition dominated by the CO-sGC 1 form with ν Fe-CO and ν CO at 488 and 1969 cm-1 , respectively, following the addition of a xenobiotic, YC-1. Further addition of a substrate, GTP, completed the change. GDP and cGMP had a significantly weaker effect, while a substrate analogue, GTP-γ-S, had an effect similar to that of GTP. In contrast, ATP had a reverse effect, and suppressed the effects of YC-1 and GTP. In the presence of both YC-1 and GTP, vinyl vibrations of heme were significantly influenced. New CO isotope-sensitive bands were observed at 521, 488, 363, and 227 cm-1. The 521 cm-1 band was assigned to the five-coordinate (5c) species from the model compound studies using ferrous iron protoporphyrin IX in CTAB micelles. Distinct from the 472 cm-1 species, both the 488 and 521 cm-1 species were apparently un-photodissociable when an ordinary Raman spinning cell was used, indicating rapid recombination of photodissociated CO. On the basis of these findings, binding of YC-1 to the heme pocket is proposed.
2011
Soluble guanylate cyclase is a dimeric ( αβ ) enzyme catalyzing the conversion of GTP to cyclic GMP which acts as a second messenger in cellular signaling. It is the only known physiological receptor of NO and binding of NO to its heme, which is covalently bound via a conserved His-β105, activates this enzyme several hundred folds over its basal level. It is known that NO-binding causes the cleavage of Fe-His bond. CO marginally activates sGC, and in the presence of some activator molecules like YC-1 and BAY it activates to the same level as NO-bound sGC, although a mechanism of this synergistic effect is hardly understood. Herein, we present evidences for the presence of two forms of CO-bound sGC in the presence of activators and deduce their structural differences and population on the basis of resonance Raman spectroscopy. A mechanism for the synergetic effect has been discussed.
Interaction of Soluble Guanylate Cyclase with YC-1: Kinetic and Resonance Raman Studies †
Biochemistry, 2000
The enzyme-soluble guanylate cyclase (sGC), which converts GTP to cGMP, is a receptor for the signaling agent nitric oxide (NO). YC-1, a synthetic benzylindazole derivative, has been shown to activate sGC in an NO-independent fashion. In the presence of carbon monoxide (CO), which by itself activates sGC approximately 5-fold, YC-1 activates sGC to a level comparable to stimulation by NO alone. We have used kinetic analyses and resonance Raman spectroscopy (RR) to investigate the interaction of YC-1 and CO with guanylate cyclase. In the presence of CO and 200 µM YC-1, the V max /K m GTP increases 226-fold. While YC-1 does not perturb the RR spectrum of the ferrous form of baculovirus/Sf9 cell expressed sGC, it induces a shift in the Fe-CO stretching frequency for the CO-bound form from 474 to 492 cm-1. Similarly, YC-1 has no effect on the RR spectrum of ferrous 1 1-385 , the isolated sGC heme-binding domain, but shifts the ν(Fe-CO) of CO-1 1-385 from 478 to 491 cm-1 , indicating that YC-1 binds in heme-binding region of sGC. In addition, the CO-bound forms of sGC and 1 1-385 in the presence of YC-1 lie on the ν(Fe-CO) vs ν(C-O) correlation curve for proximal ligands with imidazole character, which suggests that histidine remains the heme proximal ligand in the presence of YC-1. Interestingly, YC-1 does not shift ν(Fe-CO) for the CO-bound form of H105G(Im), the imidazole-rescued heme ligand mutant of 1 1-385. The data are consistent with binding of CO and YC-1 to the sGC hemebinding domain leading to conformational changes that give rise to an increase in catalytic turnover and a change in the electrostatic environment of the heme pocket.
Biochemistry, 2012
Soluble guanylate cyclase (sGC) is a hemecontaining enzyme that senses nitric oxide (NO). Formation of a heme Fe−NO complex is essential to sGC activation, and several spectroscopic techniques, including electron paramagnetic resonance (EPR) spectroscopy, have been aimed at elucidating the active enzyme conformation. Of these, only EPR spectra (X-band ∼9.6 GHz) have shown differences between low-and high-activity Fe−NO states, and these states are modeled in two different heme domain truncations of sGC, β1(1−194) and β2(1−217), respectively (Derbyshire et al., Biochemistry 2008, 47, 3892−3899). The EPR signal of the lowactivity sGC Fe−NO complex exhibits a broad lineshape that has been interpreted as resulting from site-to-site inhomogeneity, and simulated using g strain, a continuous distribution about the principal values of a given g tensor. This approach, however, fails to account for visible features in the X-band EPR spectra as well as the g anisotropy observed at higher microwave frequencies. Herein we analyze X-, Q-, and D-band EPR spectra and show that both the broad lineshape and the spectral structure of the sGC EPR signal at multiple microwave frequencies can be simulated successfully with a superposition of only two distinct g tensors. These tensors represent different populations that likely differ in Fe−NO bond angle, hydrogen bonding, or the geometry of the amino acid residues. One of these conformations can be linked to a form of the enzyme with higher activity. S oluble guanylate cyclase (sGC) catalyzes the formation of cyclic guanylate monophosphate (cGMP) from guanylate triphosphate (GTP). The synthesized cGMP is a secondary messenger for, and a critical step in, neuronal signaling, platelet aggregation, and vasodilation in mammals. 1−6 Binding of the radical diatomic gas nitric oxide (NO) to the heme cofactor is a key determinant of enzyme activation. sGC is a heterodimer that exists primarily of α1β1 subunits. 7 The β-subunit contains a heme-nitric oxide/oxygen binding (H−NOX) domain at the N-terminus, and a catalytic domain at the C-terminus. The αsubunit also contains a C-terminal catalytic domain, but does not bind heme. The heme cofactor in the β subunit is ligated by histidine, like in most globins, but it does not bind oxygen, and is stable in the ferrous-heme state. Upon NO binding, a sixcoordinate intermediate conformation forms until the Fe−His bond breaks, producing a five-coordinate Fe II −NO complex. 8−13 The α1β1 sGC five-coordinate NO complex is known to exhibit low-activity in the presence of stoichiometric NO and GTP, and high-activity in the presence of the small molecule activator YC-1 or excess NO and GTP. 14,15 In addition to the ubiquitously expressed α1 and β1 subunits, a β2 subunit exists which is expressed largely in the kidney. N-terminal truncations of both β1 and β2 have been prepared and shown to bind heme and NO. 16
Biochemistry, 2010
Modulation of soluble guanylate cyclase (sGC) activity by nitric oxide (NO) involves two distinct steps. Low level activation of sGC is achieved by the stoichiometric binding of NO (1-NO) to the heme cofactor, while much higher activation is achieved by the binding of additional NO (xsNO) at a non-heme site. Addition of the allosteric activator YC-1 to the 1-NO form leads to activity comparable to xsNO state. In this study the mechanisms of sGC activation were investigated using electronic absorption and resonance Raman (RR) spectroscopic methods. RR spectroscopy confirmed that the 1-NO form contains 5-coordinate NO-heme and showed that the addition of NO to the 1-NO form has no significant effect on the spectrum. In contrast, addition of YC-1 to either the 1-NO or xsNO forms alters the RR spectrum significantly, indicating a protein-induced change in the heme geometry. This change in the heme geometry was also observed when BAY 41-2272 was added to the xsNO form. Bands assigned to bending and stretching motions of the vinyl and propionate substituents change intensity in a pattern suggesting altered tilting of the pyrrole rings to which they are attached. In addition, the N-O stretching frequency increases, with no change in the Fe-NO frequency, an effect modeled via DFT calculations as resulting from a small opening of the Fe-N-O angle. These spectral differences demonstrate different mechanisms of activation by synthetic activators, such as YC-1 and BAY 41-2272, and excess NO. † This work was supported financially by NIH grants GM033576 (TGS) and GM077365 (MAM) Supporting Information Available. Complete reference 23 ; Mulliken charges calculated for 5-coordinate (NO)Fe(II)P under varying Fe-NO angle; RR spectra of full-length WT sGC containing one NO (1-NO), excess NO (xsNO), and the 14 NO-15 NO difference bands, covering the ν 4 and ν 7 regions; RR spectra of β1(1-385) in the presence of 1-NO, xsNO and YC-1. This material is available free of charge via the Internet at
Probing Soluble Guanylate Cyclase Activation by CO and YC-1 Using Resonance Raman Spectroscopy
Biochemistry, 2010
Soluble guanylate cyclase (sGC) is weakly activated by CO but is significantly activated by the binding of YC-1 to the sGC-CO complex. In this report resonance Raman (RR) spectroscopy was used to study selected sGC variants. Addition of YC-1 to the sGC-CO complex alters the intensity pattern of RR bands assigned to the vinyl and propionate heme substituents, suggesting changes in the tilting of the pyrrole rings to which they are attached. YC-1 also shifts the RR intensity of the ν FeC and ν CO bands from 473 and 1985 cm −1 to 487 and 1969 cm −1, respectively, and induces an additional ν FeC band, at 521 cm −1 , assigned to 5-coordinate heme-CO. Site-directed variants in the proximal heme pocket (P118A) or in the distal heme pocket (V5Y and I149Y) reduce the extent of YC-1 activation, along with the 473 cm −1 band intensity. These lower activity sGC variants display another ν FeC band at 493 cm −1 which is insensitive to YC-1 addition and is attributed to protein that cannot be activated by the allosteric activator. The results are consistent with a model in which YC-1 binding to sGC-CO results in a conformational change that activates the protein. Specifically, YC-1 binding alters the heme geometry via peripheral non-bonded contacts, and also relieves an intrinsic electronic effect that diminishes FeCO backbonding in the native, YC-1 responsive protein. This electronic effect might involve neutralization of the heme propionates via H-bond contacts, or negative polarization by a distal cysteine residue. YC-1 binding also strains the Fe-histidine bond, leading to a population of 5-coordinate sGC-CO in addition to a conformationally distinct population of 6-coordinate sGC-CO. The loss of YC-1 activation in the sGC variants might involve a weakening of the heme-protein contacts which are thought to be critical to a YC-1-induced conformational change.
Biochemistry, 1996
The soluble form of guanylate cyclase (sGC) is a hemoprotein which serves as the only known receptor for the signaling agent nitric oxide (• NO). The enzyme is a heterodimer in which each subunit binds 1 equiv of 5-coordinate high-spin type b heme. • NO increases the V max of sGC up to 400-fold by binding to the heme to form a 5-coordinate ferrous nitrosyl complex. The electron paramagnetic resonance spectrum of the ferric form of the enzyme has been obtained. The spectrum displays rhombic symmetry and is indicative of a high-spin heme. Computer simulation of the EPR spectrum yields g values of 6.36, 5.16, and 2.0 with linewidths of 3.3, 4.1, and 3.3 mT, respectively. Using electronic absorption spectroscopy, it was observed that the ferric heme binds cyanide to form a 6-coordinate low-spin complex. The rate constants for association (k on) and dissociation (k off) of cyanide at 10°C have been determined to be (7.8 (0.3) × 10-2 M-1 s-1 and (7.2 (0.2) × 10-5 s-1 , respectively. Unlike the ferrous form of the enzyme, which has a low affinity for ligands that form 6-coordinate complexes due to an unusually fast off-rate, the ferric form of the enzyme appears to have a low affinity for ligands due to a slow on-rate. The ferric heme binds azide with a K d of 26 (4 mM to form a high-spin complex. The ferric form of the enzyme has a specific activity of ∼57% that of the nonactivated ferrous form of the enzyme. However, in contrast to the mild activation of the ferrous enzyme by carbon monoxide, the ferric enzyme is not activated by cyanide. These results indicate that there may be a significant structural change in the protein upon the oxidation of the heme iron.
Journal of the American Chemical Society, 1997
Activation of soluble guanylyl cyclase (sGC) by NO correlates with scission of the proximal iron-histidine bond, as demonstrated by the application of electronic absorption and resonance Raman spectroscopy to the study of metalloporphyrin-substituted enzymes. The non-native metalloporphyrins, Mn(II)PPIX and Co(II)PPIX, can be introduced into heme-deficient sGC forming five-coordinate complexes. The similarity among Mn(II)sGC, Co(II)-sGC, and the corresponding metalloporphyrin-substituted derivatives of Mb and Hb provides confirming evidence for the presence of an axial histidine ligand in sGC. Upon addition of NO, Mn(II)sGC forms a six-coordinate species with the histidine ligand still bound to the Mn, and the enzyme is not activated. In contrast, the Co(II)-sGC(NO) adduct is five-coordinate and the enzyme is activated. These data imply that the activated state of sGC is attained when the proximal histidine-metal bond is broken.
Biochemistry, 2008
Soluble guanylate cyclase (sGC), a hemoprotein, is the primary nitric oxide (NO) receptor in higher eukaryotes. The binding of NO to sGC leads to the formation of a 5-coordinate ferrous-nitrosyl complex, and a several hundred-fold increase in cGMP synthesis. NO activation of sGC is influenced by GTP and the allosteric activators YC-1 and BAY 41-2272. Electron paramagnetic resonance (EPR) spectroscopy shows that the spectrum of the sGC ferrous-nitrosyl complex shifts in the presence of YC-1, BAY 41-2272, or GTP in the presence of excess NO relative to the heme. These molecules shift the EPR signal from one characterized by g 1 = 2.083, g 2 = 2.036, g 3 = 2.012 to a signal characterized by g 1 = 2.106, g 2 = 2.029, g 3 = 2.010. The truncated heme domain constructs β1(1-194) and β2(1-217) were compared to the full-length enzyme. The EPR spectrum of the β2 (1-217)-NO complex is characterized by g 1 = 2.106, g 2 = 2.025, g 3 = 2.010 indicating the protein is a good model for the sGC-NO complex in the presence of the activators, while the β1(1-194)-NO complex resembles the EPR spectrum of sGC in the absence of the activators. Low-temperature resonance Raman spectra of the β1(1-194)-NO and β2(1-217)-NO complexes show that the Fe-NO stretching vibration of the β2(1-217)-NO complex (535 cm −1 ) is significantly different than that of the β1(1-194)-NO complex (527 cm −1 ). This shows that sGC can adopt different 5-coordinate ferrous nitrosyl conformations, and suggests that the Fe-NO conformation characterized by this unique EPR signal and Fe-NO stretching vibration represents a highly active sGC state. † Funding was provided by NIH grants GM077365 (M.A.M.), GM73789